PROJECT SUMMARY Patients with early and locally advanced breast cancer (ELABC) are treated with a combination of pre- operative (neoadjuvant) therapy, surgical resection and post-operative (adjuvant) therapy. In recent years, overall survival for ELABC has improved to ~90% at 5 years. However, to achieve this goal, patients are often over-treated due to lack of effective biomarkers. For example, a majority of patients who receive adjuvant therapy are already cured after surgery and derive no further benefit. Similarly, almost a third of patients treated with neoadjuvant therapy are found to have pathological Complete Response upon surgery (no evidence of tumor upon histopathology), suggesting that the surgery could have been safely omitted. An effective biomarker for minimal residual disease (MRD) can help personalize treatment plans and reduce over- treatment, while preserving outcomes in patients with breast cancer. Recent literature shows post-operative detection of circulating tumor DNA (ctDNA) can identify recurrence, months before it is recognized on imaging. However, due to lack of sensitivity of current methods and limited blood sample volumes, ctDNA signal often drops below limit of detection. Current approaches for ctDNA analysis do not have adequate sensitivity to detect residual disease during and after completion of treatment. To address this gap, we have developed TARgeted DIgital Sequencing (TARDIS), an approach for multiplexed analysis of several patient-specific mutations in ctDNA. In this application, we propose to first analytically validate TARDIS for ctDNA detection and then clinically validate this approach as a biomarker for treatment monitoring and residual disease detection in early and locally advanced breast cancer. In the UH2 analytical validation phase, we will assess analytical sensitivity, specificity, accuracy, precision and reproducibility of TARDIS using reference material, tumor cell line dilutions and plasma samples from patients with breast cancer. In the UH3 clinical validation phase, we will measure baseline ctDNA detection rates in patients with early and locally advanced breast cancer, evaluate whether ctDNA levels after neoadjuvant treatment are predictive of pathological Complete Response and evaluate the prognostic value of ctDNA detection after surgical resection for disease-free survival. Our goal is to enable greater precision in treatment of patients with early and locally advanced breast cancer. Once validated, ctDNA analysis and monitoring will complement existing diagnostic approaches such as imaging and histopathology to help optimize management of patients with early and locally advanced breast cancer.